The present invention relates to a medium allowing the preservation and cryopreservation of biological materials such as animal cells and viral particles, which is directly injectable or reinjectable into an organism. It relates more particularly to a medium for the preservation of biological material comprising a saline solution, modified fluid gelatin and human serum albumin.
In cell therapy or gene therapy as well as in blood transfusion or bone marrow transplantation, one of the principal problems encountered is that of the preservation of biological material. It is indeed important to be able to preserve biological material, under good conditions of viability, for a sufficiently long period of time compatible with industrial scale production and storage and also to make it possible to carry out certain tests. The most commonly used method of preservation consists in freezing the said material. However, during the freezing of cells, for example, they undergo a very high stress due in particular to a phenomenon of exosmosis and to the formation of ice inside the cell. These phenomena cause numerous cell lyses both during freezing and during thawing. The capacity to divide or to differentiate of even the non-lysed cells may be irreversibly impaired. It is very important to be able to obtain a high viability level after thawing for cells which have to be injected into a living organism (blood transfusion, bone marrow transplantation or cell therapy for example), it is indeed futile to reinject dead or damaged cells. Likewise, when cells have to be recultured, it is equally important that the viability level is high. Up until now, a cryoprotectant which prevented cell lysis was added to the medium. The protecting agent most widely used and which gives the best results is a non-injectable preservative, dimethyl sulphoxide or DMSO. DMSO becomes inserted into the cell membranes and makes it possible to stabilize them. It thus prevents the destruction of the cells. A system such as this makes it possible, at the time of thawing, to have a large number of live and viable cells which are capable of dividing. However, this method poses a major problem when the said cells have to be introduced or reintroduced into an organism. Indeed, DMSO is a product which is toxic to the cells at room temperature. DMSO permeabilizes the membranes, causing the death of the cell. It should therefore be removed before being able to reimplant the cells in the case of an autograft of modified cells or to implant them in the case of a heterograft, for example, of bone marrow cells or alternatively to carry out transfusion in the case of blood cells. The elimination of the DMSO is achieved, after thawing, generally by diluting the sample in ten volumes of DMSO-free medium, followed by centrifugation and elimination of the supernatant. This operation is repeated several times until practically all the DMSO is eliminated. This treatment involves a loss of time and, by multiplying the manipulations, increases the risks of contamination by external pathogenic agents and of losses of the said biological material.
In the presence of DMSO, the percentage of viable cells after thawing is greater than seventy per cent under optimum freezing/thawing conditions as defined later. Without DMSO, this percentage is less than twenty per cent. Up until now, freezing in the presence of DMSO was the most effective and therefore the most widely used method.